After 3-day culture in the medium not containing
>hygromycin B, the selection was begun. The problem is that all cells die
>within a 2-week period of selection. The detailed conditions of
>electroporation/selection are as follows:
After culturing for 3 days,
>selection for hygromycin B-resistant cells was begun with hygromycin B at
>150 microgram/ml.
>>I would greatly appreciate hearing your suggestions/comments.
How do you know that this is the apropriate [hyg] to use? Did you do a kill
curve to determine the least amount of hygromycin to get selection? 150ug/ml
sounds about right for many cells, but you might want to check empirically,
since your level of expression may not be high enough for a whopping dose
in your cell type.
Brett Lindenbach
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu
"I own my own pet virus. I get to pet and name her." - Cobain